SPA-IgG复合体分子内的蛋白质多靶标并行检测

通过模仿葡萄球菌干扰免疫调理的分子机制，结合蛋白质重组技术，在固相基体表面构建层层堆积的三维抗体探针，首次提出在单个复合体分子内实现蛋白质多靶标并行检测的新方法。首先在SPA两端非对称引入HisTag组氨酸标签，以反向平行的方式逐层组装5种IgG抗体，SPA-IgG复合体经镍离子螯合作用固相化。再配以相应的荧光标记二抗，与5种蛋白质抗原靶标形成双抗体夹心免疫复合物，最终对其进行全内反射单分子荧光成像。拟采用Pull-down、XRD、QCM及ELISA等手段，结合分子动力学模拟全方位分析SPA与IgG的相互作用机制、层层组装的时空规律、功能位点的空间取向及与之关联的免疫响应，由此探明蛋白质复合体的界面生物过程、构效关系及其干预路径。相关工作将为多指标生物大分子的固定与识别、功能性基因分析、重大疾病的早期诊断、蛋白质及其复合体动态行为的人工干预、不对称分子器件的设计等基础研究提供一条新思路。

In this proposal, we present the first description of an innovative approach for parallel detection of multi-target proteins within a macromolecular complex based on 3D (three-dimensional) antibody probes which are stacked on solid matrix surface layer-by-layer. It mainly imitates the molecular mechanisms of immunomodulatory interference by Staphylococcus aures, being also involved in recombinant protein technology. Firstly, two anti-parallel HisTag-SPA (polyhistidine-tagged Staphylococcal protein A) fusion proteins, obtained from differentiated introduction of HisTag to SPA, are used for the assembly of five different IgG (Immunoglobulin G) antibodies one by one. Consequently, the SPA-IgG complex, which is immobilized following the chelation of HisTag with Nickel(Ⅱ), is built up. Coupled with corresponding fluorescently labeled secondary antibodies, five different target protein antigens are captured as a double antibody "sandwich" immune complex which ultimately can be imaged by using single-molecule TIRFM (total internal reflection fluorescence microscopy). Pull-down, crystal XRD (X-ray diffraction), QCM (quartz crystal microbalance) and ELISA (enzyme-linked immunosorbent assay) or other techniques, combined with molecular dynamics simulation, are used as a tool to carry out all-round, multi-angle analysis on crucial issues such as molecular mechanism of the interaction of SPA fusion protein with IgG antibody, spatial and temporal variation of multi-layer assembly, 3D orientation of functional sites and the associated immune response. And as a result, it will help us ascertain the interfacial biological processes of protein complex, structure-activity relationships, and track its mechanism for seeking feasibility of intervention. The related work products can be applied to the immobilization or identification of multi-target macromolecules, functional gene analysis, early diagnosis of serious disease,etc., and may offer a unique idea for artificial interference with the dynamic behaviors of proteins or their complexes, intended design of asymmetric molecular devices, and other foundational researches.